Recombinant Fibrinogen

ABSTRACT

The present invention relates to nucleotide sequences encoding a fibrinogen alpha, beta or gamma chain. The sequences are optimized for expression in a eukaryotic cell culture system. Such optimized nucleotide sequences allow for the efficient expression of recombinant fibrinogen and variants thereof in intact form in a eukaryotic cell culture system.

TECHNICAL FIELD

The present invention relates to recombinant fibrinogen, to methods forproducing it at high levels in mammalian cells and to its applications.

BACKGROUND ART

Fibrinogen is a soluble plasma glycoprotein which is synthesized in thehuman body primarily by liver parenchymal cells. It is a dimericmolecule, consisting of two pairs of three polypeptide chains designatedAα, Bβ and γ, which are connected by disulfide bridges. The threepolypeptide chains are encoded by three separate genes. The wild-type Aαchain is synthesized as a 625 amino acid precursor and is present inplasma as a 610 amino acids protein, the Bβ contains 461 and the γ chain411 amino acids. The three polypeptides are synthesized individuallyfrom 3 mRNAs. Assembly of the three component chains (Aα, Bβ, and γ)into its final form as a six-chain dimer (Aα, Bβ, γ)2 occurs in thelumen of the endoplasmic reticulum (ER).

Fibrinogen circulates in blood at high concentrations (1-2 g/L) anddemonstrates a high degree of heterogeneity. Variations arise throughgenetic polymorphisms, differences in glycosylation andphosphorylations, (partial) proteolysis of the carboxy-terminal part ofthe Aα chain and alternative splicing (for review see De Maat andVerschuur (2005) Curr. Opin. Hematol. 12, 377; Laurens et al. (2006) J.Thromb Haemost. 4, 932; Henschen-Edman (2001) Ann. N.Y. Acad. Sci. USA936, 580). It is estimated that in each individual about one milliondifferent fibrinogen molecules circulate. Most of these variants, whichaccount for just a small portion of the total fibrinogen (in most casesnot more than a few percents), differ in function and structure.Proteolysis of the carboxy-terminal part of the Aα chain results inthree major circulating forms of fibrinogen having clearly differentmolecular weights. Fibrinogen is synthesized in the high-molecularweight form (HMW; molecular weight 340 kDa; the predominant form of Aαchains in the circulation contains 610 amino acids). The degradation ofone of the Aα chains gives the low-molecular weight form (LMW; MW=305kDa); the LMW′ form (270 kDa) is the variant where both Aα chains arepartially degraded at the carboxy-terminus. In normal blood, 50-70% ofthe fibrinogen is HMW, 20-50% is fibrinogen with one or two degraded Aαchains (de Maat and Verschuur (2005) Curr. Opin. Hematol. 12, 377). TheHMW and LMW′ variants show distinct differences in clotting time andfibrin polymer structure (Hasegawa N, Sasaki S. (1990) Thromb. Res. 57,183).

Well-known variants which are the result of alternative splicing are theso-called γ′ variant and the Fib420 variant.

The γ′ variant represents about 8% of the total of γ-chains. It consistsof 427 amino acids rather than 411 for the most abundant γ-chain; thefour C-terminal amino acids (AGDV) are replaced by 20 amino acids thatcontain 2 sulphated tyrosines. The fibrinogen γ′ chain is not able tobind to the platelet fibrinogen receptor IIbβ3, which is critical inregulating platelet aggregation.

The Fib420 variant, which has a molecular weight of 420 kDa, accountsfor 1-3% of the total circulating fibrinogen (de Maat and Verschuur(2005) Curr. Opin. Hematol. 12, 377). Through alternative splicing, anextra open reading frame is included at the C-terminus of the Aα-chain,thereby extending it with 237 amino acids. The additional amino acidsform a nodular structure.

Plasma derived fibrinogen is an important component of marketed fibrinsealants which are clinically applied during surgical interventions tostop bleeding and to decrease blood and fluid loss. In addition it isused to facilitate tissue adherence by using the agglutination propertyof fibrin and to improve wound healing. Fibrinogen is also usedclinically to supplement fibrinogen deficiency in hereditaryfibrinogenemia patients and in patients with an acquired fibrinogendeficiency. Intravenous administration of high dosage of fibrinogen(3-10 gram) has demonstrated to normalize clotting of blood and arrestor prevent serious bleeding in various clinical situations.

Recombinant production of human fibrinogen, be it in wild-type (HMW)format or as a variant (e.g as Fib420), has many advantages over the useof plasma derived materials. These include its preferred safety profile,the possibility to make variants in a pure way and unlimited supply.However, in order to produce it in an economically feasible way, highexpression levels of intact, functional fibrinogen are required. Inaddition, for specific applications (e.g. use of fibrinogen as anintravenous (IV) hemostat) proper post-translational modifications (e.g.glycosylation) are required.

Because of the post-translational modifications, expression in mammaliansystems is preferred. Therefore, biologically active recombinantfibrinogen has been expressed in various cells, such as baby hamsterkidney (BHK) (e.g. Farrell et al. (1991) Biochemistry 30, 9414), ChineseHamster Ovary (CHO) cells (e.g. Lord, (U.S. Pat. No. 6,037,457), Binnieet al. (1993) Biochemistry 32, 107), or African Green Monkey derived COScells (e.g. Roy et al. (1991) J. Biol. Chem. 266, 4758). However, theexpression levels are only around 1-15 μg/ml and considered inadequateto replace the large amounts of plasma fibrinogen needed in clinicalpractice. In addition, expression of human fibrinogen in yeast P.pastoris yielded 8 μg/ml, which is also not adequate for commercialmanufacturing (Tojo et al. (2008) Prot. Expr. and Purif. 59, 289).

In EP 1 661 989 it is reported that yields of at least 100 mg/L areneeded for commercial viable production. In this application levels ofup to 631.5 mg/L by CHO cells in a spinner flask are reported. However,in order to reach such levels, cells have to co-express the baculovirusP35 anti-apoptosis protein, and methotrexate, an anti-metabolite, has tobe used for amplification of the vectors. Cell densities are relativelylow (maximum in spinner flask 9.4×10⁵ cells/ml in 15 days) as comparedto what is standard in the industry e.g. Wurm (Nature Biotechnol. (2004)22, 1393) reports routine cell densities of 2×10⁶ cells/ml in 3-4 daysof subcultivation).

The most important issue for the successful production of recombinantfibrinogen is how to make enough intact, properly assembled,biologically active product at high purity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a nucleotide sequence encoding afibrinogen alpha, beta or gamma chain which is optimized for expressionin a eukaryotic cell culture system. An optimized nucleotide sequenceaccording to the invention allows for the efficient expression ofrecombinant fibrinogen in intact form in a eukaryotic cell culturesystem. The protein sequence encoded by the optimized nucleotidesequence is identical to the protein sequence encoded by thecorresponding non-optimized nucleotide sequence.

In the context of the present invention, the term ‘fibrinogen’ may referto any of the forms of fibrinogen and includes variants which havearisen through genetic polymorphisms, differences in glycosylation andphosphorylations, (partial) proteolysis of the carboxy-terminal part ofthe Aα chain and alternative splicing. In the context of the presentinvention, the terms ‘alpha chain’ and ‘Aα chain’ are usedinterchangeably. They may refer to both wild type and variants of thealpha chain, including a fibrinogen alpha chain of 644 amino acidscontaining a signal sequence (SEQ ID No. 8), a precursor fibrinogenalpha chain of 625 amino acids without signal sequence (amino acids 20to 644 of SEQ ID NO. 8), a truncated fibrinogen alpha chain of 610 aminoacids (amino acids 20 to 629 of SEQ ID NO. 8) as found in circulationand a Fib420 variant alpha chain of 866 amino acids containing a signalsequence (SEQ ID NO. 11) or without a signal sequence (amino acids20-866 of SEQ ID NO. 11).

In the context of the present invention, the terms ‘beta chain’ and ‘Bβchain’ are used interchangeably. They may refer to both wild type andvariants of the beta chain, including a fibrinogen beta chain of 491amino acids containing a signal sequence (SEQ ID No. 9) and a fibrinogenbeta chain of 461 amino acids without signal sequence (amino acids 31 to491 of SEQ ID NO. 9).

In the context of the present invention, the term ‘gamma chain’ and ‘γchain’ are used interchangeably. They may refer to both wild type andvariants of the gamma chain, including a fibrinogen gamma chain of 437amino acids containing a signal sequence (SEQ ID No. 10), a fibrinogengamma chain of 411 amino acids without signal sequence (amino acids 27to 437 of SEQ ID NO. 10), a fibrinogen gamma chain of 453 amino acids,which is the gamma-prime chain with signal sequence (SEQ ID No. 13) anda fibrinogen gamma chain of 427 amino acids, which is the gamma-primechain without signal sequence (amino acids 27 to 453 of SEQ ID NO. 13).

In the context of the present invention, fibrinogen or a fibrinogenchain is ‘in intact form’ when the amino acid sequence contains all theamino acids which were encoded for by the nucleotide sequence,optionally without the amino acids which are removed during normal cell(secretion) processing. Therefore, alpha chains having 644, 625 or 610amino acids are examples of an alpha chain in intact form.

The optimized nucleotide sequences according to the invention have a GCcontent of at least 55%, preferably of at least 58%, more preferably ofat least 60 or 65%. In one embodiment, the optimized nucleotidesequences according to the invention have a GC content in the range ofabout 55 to 70%. In another embodiment, the optimized nucleotidesequences according to the invention have a GC content in the range ofabout 60 to 65%.

The optimized nucleotide sequences of the invention encoding afibrinogen alpha, beta and gamma chain are optimized for expression in aeukaryotic cell culture system. Preferably, they are optimized forexpression in a mammalian cell culture system, such as for expression ina COS cell, BHK cell, NS0 cell, Sp2/0 cell, CHO cell, a PER.C6 cell, aHEK293 cell or insect cell culture system. More preferably, thenucleotide sequences are optimized for expression in a human cellculture system, such as for a PER.C6 cell or a HEK293 cell culturesystem.

The optimization according to the invention has a codon adaptation indexof at least 0.90, preferably of at least 0.95, more preferably of atleast 0.97. In one embodiment, a nucleotide sequence according to theinvention is optimized by codon usage adaptation to CHO cells with acodon adaption index of at least 0.95.

Nucleotide sequences according to the invention may be encoding any typeof fibrinogen chains. Preferably they are encoding mammalian fibrinogenchains, more preferably they are encoding primate fibrinogen chains,most preferably they are encoding human fibrinogen chains. Alsocombinations are possible, such as for example one or two mammalianfibrinogen chains combined with two or one rodent fibrinogen chains. Thenucleotide sequence which is optimized may be DNA or RNA. Preferably, itis cDNA.

An optimized nucleotide sequence according to the invention encoding afibrinogen alpha, beta or gamma chain shows at least 70% identity to itsrespective non-optimized counterpart. In one embodiment, an optimizednucleotide sequence of the invention encoding a fibrinogen alpha, betaand gamma chain shows 70-80% identity to its respective non-optimizedsequences. Preferably, the optimized nucleotide sequences of theinvention encoding a fibrinogen alpha, beta or gamma chain contain nocis-acting sites, such as splice sites and poly(A) signals.

An optimized nucleotide sequence according to the invention whichencodes a fibrinogen alpha chain contains no 39 basepair direct repeatsequences which are normally present in the gene encoding the alphachain of human fibrinogen. In an optimized nucleotide sequence accordingto the invention which encodes an alpha chain, the repeating sequence ischanged without changing the encoded protein sequence.

In a preferred embodiment, an optimized nucleotide sequence according tothe invention which encodes an alpha chain comprises a sequenceaccording to SEQ ID No. 4 or 7. Nucleotide sequences which encode afibrinogen alpha chain and which comprise part of these sequences arealso encompassed by the present invention. In one embodiment, anoptimized nucleotide sequence according to the invention comprisesnucleotides 60-1932 of SEQ ID NO. 4. In another embodiment, an optimizednucleotide sequence according to the invention comprises nucleotides60-1887 of SEQ ID NO. 4. In yet another embodiment, an optimizednucleotide sequence according to the invention comprises nucleotides60-2598 of SEQ ID NO. 7. Also a nucleotide sequence which comprises asequence which is at least 85%, at least 87% or at least 90%, morepreferably at least 92%, at least 94%, 96%, most preferably at least 98%or at least 99% identical to SEQ iD NO. 4 or 7 and which encode afibrinogen alpha chain, for example a fibrinogen alpha chain with asequence according to SEQ ID NO. 8 or 11 or part of these sequences,such as for example as exemplified above, are encompassed by the presentinvention.

In a preferred embodiment, an optimized nucleotide sequence according tothe invention which encodes a beta chain comprises a sequence accordingto SEQ ID No. 5. Nucleotide sequences which encode a fibrinogen betachain and which comprise part of this sequence are also encompassed bythe present invention. In one embodiment, an optimized nucleotidesequence according to the invention comprises nucleotides 93-1473 of SEQID NO. 5. Also a nucleotide sequence which comprises a sequence which isat least 85%, at least 87% or at least 90%, more preferably at least92%, at least 94%, 96%, most preferably at least 98% or at least 99%identical to SEQ ID No. 5 and which encodes a fibrinogen beta chain, forexample a fibrinogen beta chain with a sequence according to SEQ ID NO.9 or part of this sequence, such as for example amino acids 31 to 491 ofSEQ ID NO. 9, are encompassed by the present invention.

In a preferred embodiment, an optimized nucleotide sequence according tothe invention which encodes a fibrinogen gamma chain comprises asequence according to SEQ ID No. 6. Nucleotide sequences which encode afibrinogen gamma chain and which comprise part of this sequence are alsoencompassed by the present invention. In one embodiment, an optimizednucleotide sequence according to the invention which encodes afibrinogen gamma chain comprises nucleotides 81-1311 of SEQ ID NO. 6.Also a nucleotide sequence which comprises a sequence which is at least85%, at least 87% or at least 90%, more preferably at least 92%, atleast 94%, 96%, most preferably at least 98% or at least 99% identicalto SEQ ID No.6 and which encodes a fibrinogen gamma chain, for example afibrinogen gamma chain with a sequence according to SEQ ID NO.10 or partof this sequence, such as for example amino acids 27 to 437 of SEQ IDNO.10, are encompassed by the present invention.

In another preferred embodiment, an optimized nucleotide sequenceaccording to the invention which encodes a fibrinogen gamma chaincomprises a sequence according to SEQ ID No. 12. Nucleotide sequenceswhich encode a fibrinogen gamma chain and which comprise part of thissequence are also encompassed by the present invention. In oneembodiment, an optimized nucleotide sequence according to the inventionwhich encodes a fibrinogen gamma chain comprises nucleotides 81-1359 ofSEQ ID NO. 12. Also a nucleotide sequence which comprises a sequencewhich is at least 85%, at least 87% or at least 90%, more preferably atleast 92%, at least 94%, 96%, most preferably at least 98% or at least99% identical to SEQ ID No.12 and which encodes a fibrinogen gammachain, for example a fibrinogen gamma chain with a sequence according toSEQ ID NO.13 or part of this sequence, such as for example amino acids27 to 453 of SEQ ID NO.13 are encompassed by the present invention.

In another aspect, the present invention relates to a nucleotideconstruct which comprises an optimized nucleotide sequence according tothe invention which encodes a fibrinogen alpha, beta or gamma chain. Thenucleotide construct may comprise regulatory sequences which influencethe expression of the fibrinogen chains, including promoters,terminators and enhancers. In one embodiment, the nucleotide constructis a vector, such as for example a cloning vector or expression vector.The nucleotide construct may also comprise a selection marker.

In another aspect, the present invention relates to a cell comprising anoptimized nucleotide sequence according to the invention encoding afibrinogen alpha, beta or gamma chain. In the cell, the nucleotidesaccording to the invention may be present as such or in a construct,such as in an expression vector or a cloning vector. The cell istypically a host cell which is used for the production of fibrinogen.The cell comprising the nucleotide sequence according to the inventionis preferably a mammalian cell. Suitable examples of mammalian cellsinclude insect cells, COS cells, BHK cells, NS0 cells, Sp2/0 cells, CHOcells, PER.C6 cells and HEK293 cells.

Cells according to the invention produce high amounts of intact,biologically active fibrinogen. The cell is typically part of a cellline. In the present context, the phrase ‘a cell or cell line producinghigh amounts of intact fibrinogen’ refers to a cell or cell line whichproduces more than 85%, preferably more than 90%, 95% or 99% of intactproducts. Preferably, this is measured over a period of 10, 20 or 30,more preferably over 40 or 50, population doublings. In the context ofthe present invention, ‘biologically active’ fibrinogen refers tofibrinogen which polymerizes into fibrin in the presence of thrombin.Such cells or cell lines are also encompassed by the present invention.In a preferred embodiment, a cell line according to the inventionproduces intact recombinant fibrinogen at levels of at least 3 picogramper cell per day, more preferably at least 4 or 5 picogram per cell perday, even more preferably at least 7 or 10 picogram per cell per day. Ina reactor with a cell density of 30×10⁶ cell/ml, 3 picogram per cell perday corresponds to 90 mg fibrinogen per liter reactor volume per day, 5picogram per cell per day corresponds to 150 mg fibrinogen per liter perday and 7 picogram per cell per day corresponds to 210 mg fibrinogen perliter per day. Preferably at least 50% of the cell population, morepreferably at least 60%, 70% or 80% of the cell population, mostpreferably at least 90%, 95% or 99% of the cell population produces atleast 3 picogram per cell per day, more preferably at least 5 picogramper cell per day, even more preferably at least 7 picogram per cell perday.

The selection of cells or cell lines which produce high amounts ofintact fibrinogen is preferably carried out without the expression ofprotease inhibitors. In one embodiment, the selection is performed usingantibodies, preferably monoclonal antibodies which bind to the intactN-terminus of the alpha chain and intact C-terminus of the alpha chain.Suitable commercially available examples of such antibodies include theY18 antibody described by Koppert et al. (1985) Blood 66, 503 and the G8antibody described by Hoegee-de Nobel et al. (1988) Thromb. Haemost.60(3) 415. Preferably, this selection is performed in a serum-freeenvironment. This method for the selection of cell lines which produceintact fibrinogen is also part of the present invention.

In another aspect, the present invention relates to a method forproducing fibrinogen in a eukaryotic cell culture system. The methodcomprises culturing a host cell or cell line according to the inventionunder conditions wherein fibrinogen is produced. Optionally, thefibrinogen produced is recovered. Optimized and non-optimized chains maybe combined. The non-optimized chains may be obtained by geneticengineering or by synthesis, and they may be from a different sourcethan the optimized chains. In one embodiment, only one chain perfibrinogen molecule is encoded by a codon optimized nucleotide sequenceaccording to the invention, while the two other chains are encoded bytwo nucleotide sequences which are not optimized. In another embodiment,two of the three fibrinogen chains per fibrinogen molecule are encodedby codon optimized nucleotide sequences according to the invention. In apreferred embodiment, all three fibrinogen chains are encoded byoptimized nucleotide sequences. In contrast to plasma derivedfibrinogen, the fibrinogen preparation produced by this method will berather homogeneous because specific fibrinogen chains are produced. Themethod allows for the production of fibrinogen preparations whichconsist for more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,preferably more than 95%, 98% or 99% of variants, which are present inplasma in only low amounts.

In another aspect, the present invention relates to the use ofnucleotide sequences according to the invention in the preparation offibrinogen for several medical applications. In one application, thenucleotide sequences according to the invention are used to preparefibrinogen for use in fibrin sealants which are clinically appliedduring surgical interventions to stop bleeding and to decrease blood andfluid loss. In another application, the nucleotide sequences accordingto the invention may be used to prepare fibrinogen to facilitate tissueadherence by using the agglutination property of fibrin and to improvewound healing. In yet another application, the nucleotide sequencesaccording to the invention may be used to prepare fibrinogen which isused clinically for the treatment of acute bleeding episodes in patientswith congenital or acquired (e.g. through hemorrhage after trauma orduring surgery) fibrinogen deficiency by intravenous administration offibrinogen. Marketed plasma derived fibrinogen preparations are Riastap(CSL Behring LLC; marketed in the US) and Haemocomplettan (CSL BehringAG; marketed in Europe). Recombinant fibrinogen preparations would haveseveral advantages over plasma derived preparations, including apreferred safety profile, unlimited supply and the possibility tomanufacture the fibrinogen variant with the preferred activity profilefor this specific indication a pure way.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Levels of expression of recombinant human fibrinogen 1, 2, 3 and6 days after transfection of CHO cells with wild-type and codonoptimized constructs encoding the Aα, Bβ and γ chain. The experiment wasdone in duplicate. (opt) optimized sequences; (wt) wild-type sequences.

FIG. 2 Levels of expression of recombinant human fibrinogen at 1, 2, 3and 6 days after transfection of CHO cells with codon optimizedconstructs encoding the Aα, Bβ and γ chain and codon optimizedconstructs (marked as Aα(opt)+Bβ(opt)+γ(opt)) with Aα-extended, Bβ and γchain (marked as Aα-ext.(opt)+Bβ(opt)+γ(opt)). The experiment was donein duplicate.

FIG. 3 Western blot analysis of culture supernatant from batch runs ofclones M21, M25 and M57, which express recombinant human fibrinogen. Thecontrol lane (contr) contains plasma derived wild-type fibrinogen (FIB3,Enzyme Research Laboratories). The arrows indicate the breakdownproducts of the Aα chain.

FIG. 4 Western blot analysis of culture supernatant from a batch run ofclone P40 expressing variant human fibrinogen (Fib420), which has anextended Aα chain. Lane 1 is a control containing plasma derivedwild-type fibrinogen (FIB3, Enzyme Research Laboratories). Lanes 2 and 3contain culture supernatant of clone P40 Aα-extended, taken at day 4 andat day 7 of a batch run, respectively.

FIG. 5 Western blot analysis of culture supernatant of PER.C6 cells thatwere transiently transfected with γ′ containing human fibrinogen(details are described in example 9). Lane 1 contains culturesupernatant of PER.C6 cells expressing recombinant γ′ fibrinogen. Lane 2is a control, containing plasma derived wild-type fibrinogen (FIB3,Enzyme Research Laboratories).

FIG. 6 Analysis of fibrinogen for N-glycosylation by PNGase F treatmentfollowed by SDS-PAGE analysis.

The lanes are loaded as follows:

MW: the Molecular Weight Marker (BenchMark, Invitrogen)

ERL FIB3: plasma derived fibrinogen (ERL), either treated with PNGase F(+) or non-treated (−) with PNGase F.PER.C6 fbg: PER.C6 derived fibrinogen, either treated with PNGase F (+)or non-treated (−) with PNGase F.2 μg of fibrinogen was loaded per lane; staining was done usingCoomassie Blue. Analysis was performed using a reduced 10% BisTris gel(NuPage, Invitrogen).

FIG. 7 ROTEM analysis: clotting time.

Clotting time was determined by ROTEM analysis. 200 μl of pooled normal(citrate) plasma or 100 μl of pooled normal (citrate) plasma mixed 1:1with Haemocomplettan (CSL Behring GmbH, Marburg, Germany) or PER.C6fibrinogen (both 2 mg/ml in TBS). CaCl₂ was added to a finalconcentration of 17 mM. To start clotting α-thrombin was added to afinal concentration of 1 IU/ml. Total reaction volume was 240 μl. Thefigure displays the clotting time (seconds) for plasma mixed 1:1 withfibrinogen:

-   -   1. Plasma derived fibrinogen (CSL Behring, Marburg, Germany)    -   2. PER.C6 derived fibrinogen

All measurements were done in duplicate.

FIG. 8 ROTEM analysis: clot firmness. Clot firmness was determined byROTEM analysis. The figure expresses the A10 value (mm), which is thefirmness of the clot at time 10 minutes, for plasma mixed 1:1 withfibrinogen. Experimental details are the same as described in the legendof FIG. 7.

-   -   1. Plasma derived fibrinogen (CSL Behring, Marburg, Germany)    -   2. PER.C6 derived fibrinogen

All measurements were done in duplicate.

FIG. 9 ROTEM analysis: clot formation time. Citrated blood from ahealthy individual was either or not diluted with Ringer's lactate(Baxter, Utrecht, The Netherlands). Subsequently, blood diluted withRinger's lactate was either or not (control) replenished with plasmaderived or recombinant fibrinogen.

The figure display the following:

-   -   1. 300 μl blood    -   2. 150 μl blood, 100 μl Ringer's lactate (RL), 50 μl TBS    -   3. 150 μl blood, 100 μl RL, 50 μl Haemocomplettan 6.5 mg/ml (1.1        mg/ml final conc.)    -   4. 150 μl blood, 100 μl RL, 50 μl recombinant hFbg 6.5 mg/ml        (1.1 mg/ml final conc.)        In all conditions 20 μl star-TEM and 20 μl ex-TEM reagent        (Pentapharm GmbH, Munich, Germany) was used to start        coagulation.        Normal range (35-160 sec) are values found for healthy        individuals. CFT values of 160-220 sec are found in patients        with normally unimpaired haemostasis but with reduced reserve.

FIG. 10 ROTEM analysis: clot firmness. Citrated blood from a healthyindividual was either or not diluted with Ringer's lactate (Baxter,Utrecht, The Netherlands). Subsequently, blood diluted with Ringer'slactate was either or not (control) replenished with plasma derived orrecombinant fibrinogen.

Measurement conditions were as follows:

-   -   1. 300 μl blood    -   2. 150 μl blood, 100 μl Ringer's lactate (RL), 50 μl TBS    -   3. 150 μl blood, 100 μl RL, 50 μl Haemocomplettan 6.5 mg/ml (1.1        mg/ml final conc.)    -   4. 150 μl blood, 100 μl RL, 50 μl recombinant hFbg 6.5 mg/ml        (1.1 mg/ml final conc.)        In all conditions 20 μl star-TEM and 20 μl ex-TEM reagent        (Pentapharm GmbH, Munich, Germany) was used to start        coagulation.        Normal range (53-72 mm) are values found for healthy patients        without coagulation disorders. MCF values of 45-40 mm found in        patients indicate a bleeding risk.

EXAMPLES Example 1 Preparation of Optimized cDNA Constructs

cDNAs coding for human fibrinogen polypeptide chains Aα, Bβ, γ,Aα-extended (Fib420) and γ′ were synthesized in both wild type (in thisExample referring to the non-optimized format) and codon optimizedformat by GeneArt (Regensburg, Germany): (i) cis-acting sites (splicesites, poly(A) signals) were removed; (ii) repeat sequence of Aα chainwas modified; (iii) GC content was increased for prolonged mRNA halflife; (iv) Codon usage was adapted to CHO (codon adaptionindex—CAI->0.95). Wildtype reference used were NM_(—)021871 for thealpha chain, NM_(—)005141 for the beta chain and NM_(—)000509 for thegamma chain.

The cDNAs coding for Aα (SEQ ID NO. 1), Bβ (SEQ ID No. 2) and γ (SEQ IDNO. 3) chain in wild type format and for optimized Aα (SEQ ID NO. 4), Bβ(SEQ ID No. 5) and γ (SEQ ID NO. 6) cDNAs were compared and the resultsare shown in Table 1. Optimized Aα-extended (Fib420) (SEQ ID NO.7) andγ′ sequences (SEQ ID NO.12) are also displayed in Table I.

Wild type alpha (SEQ ID no. 1), beta (SEQ ID NO. 2) and gamma (SEQ iDNO.3) chain cDNA and optimized alpha (SEQ ID no. 4), beta (SEQ ID no.5), and gamma (SEQ ID no. 6), chain cDNA were subcloned in pcDNA3.1deriviates. Both -wildtype and optimized-Aα-chains, and Aα-extended(Fib420) in pcDNA3.1(+) neo, both Bβ chains in pcDNA3.1(+)hygro and bothγ chains in pcDNA3.1(−)hygro (Invitrogen, Carlsbad, USA). Optimizedγ′-chain was subcloned in pcDNA3.1(+) hygro.

TABLE 1 Match Codon Adaptation Index Fibrinogen (%) (CAI) GC content (%)chain Wt/opt Wild type optimized wild-type optimized Aα chain 72 0.710.97 48 65 Bβ chain 77 0.69 0.96 45 60 γ chain 76 0.68 0.97 42 60 Fib420 Aα 72 0.71 0.98 48 63 chain γ′ chain 75 n.d. 0.97 42 60

Example 2 Transient Expression of Codon-Optimized and Wild-TypeFibrinogen Sequences in CHO Cells

To verify whether the optimized sequences improved protein expression,transient transfections were done in CHO-S cells (Invitrogen, Carlsbad,USA), according to the manufacturer's instructions. Briefly, CHO-S cellswere seeded on the day prior to transfection at 0.6×10⁶ cells/ml inFreeStyle culture medium supplemented with 8 mM L-glutamine. On the dayof transfection, cells were diluted to a concentration of 1×10⁶ cells/mlin 15 ml medium in a 125 ml shake flask (Corning Life Sciences, Corning,USA). A total of 18.75 pg expression plasmid (6.25 pg for eachindividual chain) was mixed with 0.3 ml OptiPro SFM. Subsequently 0.3 mlFreeStyle MAX Transfection Reagent (16× diluted in OptiPro SFM) wasadded and mixed gently. After a 10 minute incubation at room temperaturethe DNA-FreeStyle MAX mix was gently added to the CHO-S cells, slowlyswirling the shake flask. The experiment was performed in duplicate.

Transfected cells were incubated at 37° C., 5% CO₂ on an orbital shakerplatform rotating at 125 rpm. On day 1, 2, 3, and 6 post transfectionsamples were collected to measure recombinant fibrinogen expression.

Protein expression was measured with an ELISA specific for humanfibrinogen. Certified Maxisorb Elisa plates (Nunc, ThermofisherScientific, Roskilde, Denmark) were coated overnight with 100 μl 10μg/ml G8 monoclonal antibody (TNO KvL, Leiden, The Netherlands) raisedagainst human fibrinogen (Hoegee-de Nobel et al. (1988) Thromb. Haemost.60(3) 415) in PBS (Invitrogen) at 4° C. Then the plates were washed withPBST (PBS/0.05% Tween20 tablets, Calbiochem, EMD, San Diego, USA) and100 μl of either culture supernatant sample or fibrinogen standard wereadded. The fibrinogen standard contained fibrinogen (FIB3 HumanFibrinogen, Enzyme Research Laboratories (ERL), Swansea, UK) dissolvedand diluted in PBST at the following concentrations:100-75-50-25-12.5-6.25-3.125-0 ng/ml. Tissue culture supernatant sampleswere diluted 1:10-1:500 in PBST. After 1 hour incubation at roomtemperature the plates were washed 3 times with 200 μl PBST per well andtapped dry on a paper towel. Then 100 μl of HRP conjugated Y18monoclonal antibody (TNO KvL, Leiden, The Netherlands), diluted 1:10.000in PBST, was added. This was incubated for 1 hour at room temperature,followed by washing the plates 4 times with 200 μl PBST per well; toafter each wash step the plates were tapped dry on a paper towel. Then,100 μl TMB Ultra (Pierce, Thermofisher Scientific, Rockford, USA) wasadded to each well, followed by an incubation of 4-30 minutes at roomtemperature. The reaction was stopped by addition of 100 μl 2M H₂SO₄(Merck KGaA, Darmstadt, Germany) to each well and the OD450 wasdetermined using an ELISA plate reader.

The results are shown in FIG. 1. The data clearly show that theoptimized sequences improve the expression of fibrinogen dramatically atall time points where samples were analysed. The increase in expressionlevel for optimized constructs ranges from 7.9-10.5 times.

Example 3 Transient Expression of Codon-Optimized Fibrinogen 420 inSerum-Free Cultured CHO Cells

Transfection and analysis were performed as described in Example 2. Theextended Aα-chain cDNA sequence used in this experiment is an optimizedextended Aα sequence (SEQ ID No. 7) and codes for a secreted polypeptideof 847 amino acids (SEQ ID No. 11).

The results are shown in FIG. 2 and clearly show that the expressionlevels of a fibrinogen variant, in this case the Fib420 variant withextended Aα chains, are in the same range as the enhanced levels for theoptimized ‘wild-type’ Aα-chain variant.

Example 4 Generation of Cho Cells Stably Expressing Human Fibrinogenfrom Codon-Optimized Fibrinogen cDNAs Under Serum-Free Conditions

For the cell-line generation described in this report thesequence-optimized pcDNA3.1 derived plasmids were used, as described inExample 1. Briefly, CHO-S cells (Invitrogen) were subcultured inFreeStyle medium (Invitrogen) supplemented with 8 mM L-glutamine(Invitrogen) according to the manufacturer's instruction. Routinelycells were cultured in a 125 ml shake flask format containing 10% (v/v)culture medium (=12.5 ml). The cultures were placed in a humidifiedincubator at 37° C. and 5% CO₂ on a horizontally shaking platform at 125rpm. Transfections of CHO-S cells (invitrogen) were performed accordingto the manufacturer's instructions. After transfection the cultures wereincubated overnight in a humidified incubator at 37° C. and 5% CO₂ on ahorizontally shaking platform at 125 rpm.

The day following the transfection, the cells were counted, and seededinto 96-well plates (seeding density 200 cells/well) in FreeStyle mediumsupplemented with 8 to mM L glutamine and the selection agents Geneticin(Invitrogen) and Hygromycin B (Invitrogen) (both at a finalconcentration of 500 μg/ml: from here on “selection medium”). Culturevolume in each well was 100-200 μl. Plates were placed in a humidifiedincubator at 37° C. and 5% CO₂ under stationary conditions. The mediumwas changed twice a week with 100 μl selection medium. The plates werescreened for cell growth microscopically. After 10 days resistant clonesbecame apparent. These clones were transferred into 48 well platescontaining 500 μl selection medium.

When clones reached approximately 50% confluence the medium was sampledand stored at −20° C. until ELISA analysis for fibrinogen expressionlevels was performed (see example 2). Based on ELISA results clonespositive for fibrinogen were sub-cultured to 6-well plates. Again atapproximately 50% confluence medium of each clone was sampled andanalysed for expression by ELISA. Based on the ELISA data, selected highexpressing clones were transferred to T25 flasks and 3-5 days later toT75 cm² flasks. Then, cells were transferred to shaker cultures, wherethey were inoculated at a concentration of 0.2×10⁶ viable cells/ml in125 ml shake flasks containing 12.5 ml selection medium. Flasks wereplaced on an ELMI horizontal shaker at 125 rpm in a humidified incubatorat 37° C. and 5% CO₂. After reaching cell densities of more than 0.5×10⁶viable cells/ml, the cells were sub-cultured into a new 125 ml shakeflask with fresh medium three times a week at an inoculationconcentration of 0.2×10⁶ viable cells/ml until reproducible growthcharacteristics were established (usually within 2 weeks). Cultures ofselected clones were maintained in selection medium.

For batch testing, shaker cultures were started of the selected clonesin 12.5 ml FreeStyle medium supplemented with 8 mM L-glutamine in 125 mlshake flasks. The cultures were inoculated at 0.2×10⁶ viable cells/ml.Flasks were placed on the DOS-10-ELMI horizontal shaker at 125 rpm at37° C. and 5% CO₂ in a humidified incubator. Samples were collected onday 1, 2, 3, 4, and 7 post seeding, and total cell count and viability(by Trypan Blue staining) were determined. The samples were cleared fromcells by 300×g centrifugation and supernatants were stored at −20° C.until fibrinogen concentrations could be determined.

For Western blotting, samples containing fibrinogen were mixed with 5 μl4× concentrated NuPAGE LDS sample buffer (Invitrogen, Paisley, UK) and 2μl 10× concentrated NuPAGE sample reducing agent (Invitrogen). The finalvolume was adjusted to 20 μl with deionized water (Invitrogen/Gibco).Samples were heated for 10 minutes at 70° C. and loaded on a NuPAGENovex gel (10%; Bis-Tris Mini gel, Invitrogen), according to theinstructions of the manufacturer. The gel was run for 1 hour at 200Volt. Blotting buffer was prepared by mixing 44 ml 25× NovexTris-Glycine Transfer Buffer (Invitrogen), 836 ml of demiwater and 220ml of methanol (Merck). The solution was precooled for a minimum of 30minutes at −20° C. A piece of PVDF membrane (Pierce) is activated forabout 15 seconds in methanol. The membrane, 6 pieces of Gel BlottingPaper and 2 blotting pads were then incubated in blot buffer for a fewminutes. The membrane was placed on the gel in a blotcassette which wasput in a blotting chamber (Bio-Rad Laboratories, Hercules, USA) holdinga cold pack frozen at −20° C. Protein transfer was performed at 100Volts across the gel/membrane assembly for 1 hour,

To visualize the fibrinogen bands on the membrane, the blot wasincubated in 50 ml blocking buffer (3% Low fat milk powder (Elk,Campina, Meppel, The Netherlands) in PBS) on a platform shaker. Then,the blot was washed for 10 minutes in 50 ml washing buffer (0.05%Tween20 in PBS) and incubated for 1 hour with 10 ml of blocking buffercontaining 1/2000 dilution of the HRP conjugated monoclonal antibodyY18/PO (Koppert et al. (1985) 66, 503). Then the blot was washed 2×short (less than 1 minute), 1×15 min, and 3×5 min. in 50 ml washbuffer,followed by a 10 min incubation in 50 ml PBS.

The bands were visualized with ECL (cat#32209, Pierce). The image wascaptured using a ChemiDoc-It Imaging System (UVP, California, US).

Multiple rounds of generation of stable clones were performed. Onlyclones which produced more than 3 picogram per cell per day (pcd) in 7days batch cultures were collected. Some clones produced more than 5 pcdin 7 days batch cultures.

As indicated before, clones were tested both for fibrinogen productionas well as for quality of the produced fibrinogen. Some clones producedhigh levels of intact fibrinogen, other generated intact product butalso showed degradation products. The Aα-chain is most sensitive toproteolysis as compared to the Bβ and γ, so the primary screening toolscreening was in first instance focused on testing the Aα chain forintegrity. A typical example is shown in FIG. 3, where clone M21 clearlyshows degradation of the Aα chain, whereas M25 and M57 do not. Westernblot analysis of these samples for integrity of the Bβ and γ proved thatthese chains were still intact, even if the Aα chain showed proteolyticbreakdown.

Example 5 Generation of Stable Cell Lines Expressing Human Variant Fib420

Stable cell lines expressing a variant of human fibrinogen, viz. the Fib420 variant which has an extended Aα chain (847 amino acids rather than625) were generated. A codon optimized construct (SEQ ID No. 7) was usedand clones were generated under serum-free conditions, as described inExample 5.

The supernatant of two positive clones which produced more than 3 pcd ina 7 days batch culture were checked for intact extended Aα chain(Fib420) using Western blot analysis. It was clear (FIG. 4) that even inthe high producing clones the Aα chain of Fib420 was extended andintact.

Example 6 Generation of PER.C6 Cells Stably Expressing Human Fibrinogenfrom Codon-Optimized Fibrinogen cDNAs Under Serum-Free Conditions

PER.C6 cells (Fallaux et al. (1998) Hum. Gene Ther. 9(13) 1909) wereused as a host for the expression of recombinant human fibrinogen.Briefly, PER.C6 cells were cultured in suspension in mAb medium (SAFC,Hampshire, UK) and transfected using the AMAXA (Lonza, Cologne, Germany)nucleofection device (program A-27, using Nucleofector kit T with threevectors encoding the three different chains of the human fibrinogenprotein (Aα-, Bβ-, and γ chain) and containing the optimized cDNA chains(SEQ ID no.4, SEQ ID no.5, and SEQ ID no.6, resp).

After nucleofection, the cells were cultured for 1 day in T-flasks andsubsequently seeded in 96 well plates (Greiner, Alphen a/d Rijn, TheNetherlands) at a density of 1000-3000 cells/well. Then mAb mediumcontaining 125 μg/ml Geneticin (Invitrogen) was added. Afterapproximately three weeks, clones in about 10-30% of individual wells ofthe 96-well plate appeared, which were subsequently expanded to 48-, 24-and 6 wells plates and then subcultured into T25 and T80 culture flasks.Throughout the expansion the cultures were screened for expressionlevels of human fibrinogen. Low and non-expressing cells were discarded.Subsequently cells were cultured in shake flasks (125 ml, Corning).

In total, 579 clones were identified in 96-wells plates. Based onfibrinogen expression levels throughout expansion, 43 clones wereselected and subcultured to shake flasks. 10 out of these 43 recombinanthuman fibrinogen producing PER.C6 cell lines were selected for initialbatch testing, based on growth- and production characteristics. Batchtesting of the 6 selected PER.C6® cell lines in VPRO medium (SAFC)showed volumetric production levels up to 279 mg/L recombinant humanfibrinogen, and a specific productivity of 19.8 pcd. Finally a batchculture in VPRO medium was performed with a medium change at time ofsampling, which resulted in cumulative volumetric production levels ofup to 515 mg/L recombinant human fibrinogen.

Example 7 Generation of Stable PER.C6 Cell Lines Expressing HumanFibrinogen with an Aα-Chain of 610 Amino Acids

In order to generate an expression plasmid encoding the Aα610 of thepredominant form of plasma fibrinogen in the blood circulation, a cDNAfragment (SEQUENCE ID 8), optimized as described before, encoding aminoacids 1-610 of the Aα chain was cloned into expression plasmidpcDNA3.1(+) neo, according to standard procedures. The generation ofPER.C6 cell lines producing recombinant human fibrinogen is similar asdescribed before (see example 6). Sequences used for Aα-, Bβ-, and γchain are SEQ ID no.8, SEQ ID no.5, and SEQ ID no.6, resp.

After transfection of PER.C6 cells and plating in 96-well plates, 310clones were transferred and screened in 48-well plates. At the end ofthe expansion path, 24 out of these 310 were transferred to shakerflasks, of which after an initial batch test, 8 were selected forstability and productivity testing in batch culture.

Yields in batch culture were similar to yields obtained with cell linesthat express the Aα-chain in 625 amino acid format, clearlydemonstrating that expression of Aα chains from a cDNA coding for a 610amino form does not impair expressions levels.

Protein analysis using SDS-PAGE and Western blotting analysis indicatedthat the recombinant fibrinogen was produced in intact format.

Example 8 PER.C6 Cell Lines Expressing Recombinant Human FibrinogenBased on Extended Aα-Chain (Fib420 Variant)

The generation of PER.C6 cell lines producing recombinant humanfibrinogen is similar as described before (see examples 6 and 7). Insummary, sequences used for Aα-, Bβ-, and γ chain are SEQ ID no. 7, SEQID no.5, and SEQ ID no.6, resp.

After transfection and plating in 96-well plates, 325 clones weretransferred and screened in 48-well plates. At the end of the expansionpath, 24 clones were transferred to shaker flasks, of which 8 wereselected for stability and expression analysis in continued batchculture testing.

Yields in batch culture were similar to yields obtained with cell linesthat express the Aα-chain in 610 or 625 amino acid format, indicatingthat the extension of the Aα-chain does not impair expression levels.This was not expected on forehand, as plasma derived fibrinogen onlycontains 1-3% of extended Aα-chain as compared to 610/625 Aα-chaincontaining fibrinogen. Protein analysis using SDS-PAGE and Westernblotting analysis indicate that the recombinant fibrinogen is producedin intact format, with the α-chain having the expected size (similar toCHO produced Aα-chain from Fib420 as shown in FIG. 4).

Example 9 Transient Expression of γ′ Codon-Optimized Fibrinogen inSerum-Free Cultured CHO Cells

Transient transfection and analysis were performed as described inExample 2. The extended γ′-chain cDNA sequence used in this experimentis an optimized extended γ′ sequence (SEQ ID No. 12) and codes for apolypeptide of 453 amino acids. After removal of the signal peptide, asecreted polypeptide of 427 amino acids (amino acids 27 to 453 of SEQ IDNO. 13).

The results showed that the expression levels of the fibrinogen variantwith the γ′ chains are in the same range as the enhanced levels for theoptimized ‘wild-type’ variant. Culture supernatant was analyzed byWestern blotting analysis. The results (FIG. 5) show that γ′-chainrecombinant fibrinogen, in lane 1, runs slower than ‘wild-type’fibrinogen, in lane 2. This indicates that the γ′-chain in recombinantfibrinogen is extended as compared to γ-chain in plasma derivedfibrinogen and that the γ′-chain is intact and not degraded.

Example 10 Purification of Recombinant Human Fibrinogen

Recombinant human fibrinogen from Example 6 was purified from cellculture supernatant according to standard methods. Briefly, (NH₄)2SO₄was added to the culture supernatant to 40% saturation and theprecipitate is collected by centrifugation. Subsequently, theprecipitate was dissolved in TBS (50 mM Tris-HCl, pH7.4, 100 mM NaCl),diluted (10-fold) in loadingbuffer (5 mM Tris-HCl pH 7.4, 0.01%Tween-20, 0.5M (NH₄)2SO₄) and loaded on a HiTrap Butyl FF (20 ml) (GEHealthcare, Uppsala, Sweden) Hydrophobic Interaction Column (HIC). Boundprotein was eluted by loadingbuffer containing a gradient of (NH₄)2SO₄of 0.5-0 M (NH₄)2SO₄ in 20 column volumes. The peak fractions of the HICpurification were subjected to a buffer change by dialysis versus TMAEloading buffer (5 mM Tris-HCl pH 8.5, 0.01% Tween-20) and subsequentlyloaded on a Fractogel EMD TMAE (m) 40-90 μm (20 ml) (Merck KGaA,Darmstadt, Germany) Ion Exchange Column. Recombinant human fibrinogenwas subsequently eluted using a continuous salt gradient of 0-1 M NaClin 20 column volumes.

Recombinant human fibrinogen in the peak fractions was precipitatedagain by adding (NH₄)₂SO₄ to 40% saturation and collected bycentrifugation. Finally the material was dissolved in TBS (50 mMTris-HCl, pH7.4, 100 mM NaCl) and dialysed against TBS to remove anyremaining (NH₄)₂SO₄.

Example 11 Functionality of Recombinant Fibrinogen

Purified recombinant PER.C6 fibrinogen, as produced by cell linesgenerated in Example 6, was subjected to a number of tests to evaluateit's quality and functionality and to compare it with plasma derivedfibrinogen. N-glycosylation of fibrinogen was tested by treatment offibrinogen with PNGase F, which is an amidase that removes N-linkedcarbohydrate structures from proteins (Maley, F. et al. (1989) Anal.Biochem. 180, 195). Samples of purified fibrinogen, derived from PER.C6cultures, as well as plasma derived fibrinogen (FIB3 Human Fibrinogen,ERL) were treated with PNGase F (New England Biolabs, Ipswich, Mass.,US), according to the manufacturer's instructions.

The results (FIG. 6) indicate that PNGase F treatment results in adecreased molecular mass for the Bβ- and γ-chains, as determined bySDS-PAGE, for both plasma derived FIB3 (ERL) and PER.C6 basedfibrinogen. This is consistent with the fact that both chains containone N-glycosylation site (Henschen-Edman (2001) Ann. N.Y. Acad. Sci. USA936, 580). The data show that both pre- and post PNGase F treatment,distinct single bands are visible for both the Bβ- and γ-chain. Thisindicates that, as for plasma derived fibrinogen, all of these chains inrecombinant fibrinogen are glycosylated. The Aα-chain of humanfibrinogen contains no N-glycans, hence the molecular weight is notchanged upon PNGase F treatment. In conclusion, these data indicate thatthe N-glycosylation pattern of PER.C6 based fibrinogen is similar to theplasma derived counterpart.

Biological activity of PER.C6 derived fibrinogen was further tested in apolymerization assay, carried out as described by Koopman et al. (1992)Blood 80(8):1972. The results obtained were similar to those obtainedwith CHO-derived human fibrinogen. The assay measures the polymerizationof fibrinogen under the action of thrombin to form fibrin.Polymerization is measured by recording OD350 nm in time. Polymerisationof recombinant PER.C6 fibrinogen in plasma was equal to plasma andCHO-derived fibrinogen.

Clottability of PER.C6 derived fibrinogen, purified as described, wastested by addition of α-thrombin (7.5 IU/ml) (ERL, Swansea, UK) andCaCl2 (2 mM final concentration), followed by an incubation at 37° C.for 1 hour. The resulting clot was then collected by centrifugation inan Eppendorf vial (15 min, 5000 rpm, eppendorf centrifuge). Thesupernatant was transferred to a new tube and the clot was dissolved inalkalic urea. Protein was measured in the supernatant and the clot byA280 measurement. Fibrinogen content of the supernatant was measured byELISA (G8-Y18 antibodies). Results were similar for plasma derivedfibrinogen and PER.C6 derived fibrinogen: 97% and 94% of the protein wasmeasured in the dissolved clot (5% and 8% in the supernatant),respectively. No fibrinogen could be detected in the supernatant byELISA. These results further support biological similarity betweenplasma derived and recombinant human fibrinogen.

Clotting time and clot firmness of recombinant and plasma derivedfibrinogen were measured using ROTEM analysis. ROTEM® (Pentapharm GmbH,Munich, Germany) stands for ROtation ThromboElastoMetry. The techniqueutilizes a rotating axis submerged in a (blood) sample in a disposablecuvette. Changes in elasticity under different clotting conditionsresult in a change in the rotation of the axis, which is visualized in athromboelastogram, reflecting mechanical clot parameters (see e.g.Luddington R. J. (2005) Clin Lab Haematol. 2005 27(2):81). Pooled normal(citrate) plasma was mixed 1:1 with Haemocomplettan (CSL Behring GmbH,Marburg, Germany) or PER.C6 fibrinogen (both 2 mg/ml in TBS). CaCl₂ wasadded to a final concentration of 17 mM. To start clotting, α-thrombinwas added to a final concentration of 1 IU/ml. Clotting time andclot-firmness were analysed by ROTEM.

Diluting citrated plasma compromises both clotting time and clotfirmness. The results indicate that restoring fibrinogen levels indiluted plasma by adding purified fibrinogen restores both clotting time(FIG. 7) and clot firmness (FIG. 8) to the same extent for plasmaderived fibrinogen and recombinant fibrinogen. Similar data wereobtained with CHO based recombinant fibrinogen.

These results indicate that recombinant human fibrinogen would be a goodalternative to supplement fibrinogen deficiency in hereditaryfibrinogenemia patients and in patients with an acquired fibrinogendeficiency.

Example 12 ROTEM Analysis in Human Blood

In order to further prove that recombinant human fibrinogen can be usedfor treatment of patients with fibrinogen deficiency, experiments werecarried out in blood from a healthy human individual. Fibrinogendeficiency was mimicked by diluting the blood 1:1 with Ringer's lactate(Baxter, Utrecht, The Netherlands). Then, using ROTEM analysis asdescribed in example 11, clot formation time and clot firmness weredetermined. To restore the fibrinogen levels in blood that was diluted1:1 with Ringer's lactate, either plasma derived or recombinantfibrinogen was added.

The data (FIG. 9) indicate that clot formation time of blood dilutedwith Ringer's lactate was outside the normal range, as in a clinicalsituation for a patient that has low fibrinogen levels. Addition ofeither recombinant or plasma derived fibrinogen resulted in restorationof the clot formation time to a level within the normal range. Thisindicates the potential of recombinant fibrinogen for intra-venoustreatment of patients with low to fibrinogen levels.

When blood was diluted with Ringer's lactate maximum clot firmness (MCF)was reduced to a level associated with bleeding risk in patients (FIG.10). When fibrinogen levels were replenished with plasma derived orrecombinant fibrinogen MCF was restored to normal levels, therebyunderscoring the potential for use of recombinant fibrinogen forintra-venous treatment of patients with low fibrinogen levels. It is ofnote, that for approval of Riastap in the US, clinical efficacy wasbased on a surrogate endpoint, which was Maximum Clot Firmness measuredby Thromboelastography.

1. A nucleotide sequence encoding a fibrinogen alpha, beta or gammachain which is optimized for expression in a mammalian cell culturesystem.
 2. A nucleotide sequence according to claim 1 which is optimizedfor expression in a COS cell, a BHK cell, a NSO cell, a CHO cell, aSP2/0 or a human cell culture system.
 3. A nucleotide sequence accordingto claim 1 which is optimized for expression in a PER.C6 cell or aHEK293 cell culture system.
 4. A nucleotide sequence according to claim1 which is optimized by codon usage adaptation to CHO cells.
 5. Anucleotide sequence according to claim 1 which has a GC content of atleast 55%.
 6. A nucleotide sequence according to claim 1 which shows atleast 70% identity to its respective non-optimized counterpart.
 7. Anucleotide sequence according to claim 1 wherein the codon optimizedfibrinogen chains contain no cis-acting sites.
 8. A nucleotide sequenceaccording to claim 1 having a nucleotide sequence according to SEQ IDNo. 4 or 7, or part thereof, or a nucleotide sequence which has asequence which is at least 85% identical to SEQ ID NO. 4 or 7 and whichencodes a fibrinogen alpha chain.
 9. A nucleotide sequence according toclaim 1 having a nucleotide according to SEQ ID No. 5 or part thereof,or a nucleotide sequence which has a sequence which is at least 85%identical to SEQ ID No. 5 and which encodes a fibrinogen beta chain. 10.A nucleotide sequence according to claim 1 having an nucleotideaccording to SEQ ID No. 6 or 12, or part thereof, or a nucleotidesequence which has a sequence which is at least 85% identical to SEQ IDNo. 6 or 12 and which encodes a fibrinogen gamma chain.
 11. A nucleotideconstruct which comprises a nucleotide sequence according to claim 1.12. A cell comprising a nucleotide sequence according to claim 1 or anucleotide construct according to claim
 11. 13. A cell or cell linewhich produces intact recombinant fibrinogen at levels of at least 3picogram per cell per day.
 14. A method for the production of fibrinogenin a eukaryotic cell culture system which method comprises culturing acell according to claim 12 under conditions wherein fibrinogen isproduced, and optionally, recovering the fibrinogen produced.
 15. Afibrinogen preparation prepared by the method of claim
 14. 16. Afibrinogen preparation in which more than 10% of the alpha, beta orgamma chains are a variant type.
 17. A fibrinogen preparation accordingto claim 16 wherein the variant type is a gamma prime chain or an alphaextended chain.
 18. A fibrinogen preparation according to claim 14 foruse as a medicament, as a tissue sealant or to facilitate tissueadherence.
 19. Method of using a fibrinogen preparation according toclaim 14 for the preparation of a medicament for the treatment orprevention of fibrinogen deficiency.
 20. A fibrinogen preparationproduced by recombinant technology in which more than 85% of thefibrinogen is in the intact form.
 21. A method for the selection of acell or cell line which produces intact recombinant fibrinogen, whichmethod comprises the use of two antibodies, wherein one antibodyselectively binds to the intact N-terminus of the alpha chain and theother antibody selectively binds to the intact C-terminus of the alphachain.
 22. A nucleotide sequence according to claim 4 with a codonadaptation index of at least 0.95.
 23. A method for the production offibrinogen in a eukaryotic cell culture system which method comprisesculturing a cell or cell line according to claim 13 under conditionswherein fibrinogen is produced, and optionally, recovering thefibrinogen produced.